Reporter molecules are routinely used to monitor molecular events in the fields of biology, biochemistry, immunology, cell biology and molecular biology. For example, reporter molecules are employed in assays where the levels of the reporter molecule are due to transcription from a specific promoter linked to the reporter molecule. These assays can be used to study eukaryotic gene expression, receptor activity, transcription factors, intracellular signaling, mRNA processing, protein folding, and the like. Reporter molecules that are typically used in such assays include radioactive isotopes, fluorescent agents, enzymes, and luminescent agents. See for example, Akhavan-Tafti, et al, in: Bioluminescence and Chemiluminescence. Fundamentals and Applied Aspects. Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence. Cambridge, September 1994. Eds. Campbel, Kricka, Stanley. John Wiley and Sons 1994.
Two luminescent enzymes that are particularly useful in assay systems are firefly luciferase and Renilla reniformis luciferase. The substrates for these luciferases and the reaction products they produce are shown in FIGS. 1 and 2. The quantity of light (i.e. the number of photons) produced in the reaction, can be measured and used to calculate the concentration of luminescent enzyme in the reaction.
Firefly luciferase is a 61 kDa monomeric protein that does not require post-translational processing for enzymatic activity. Thus, it functions as a genetic reporter immediately upon translation. Photon emission is achieved through oxidation of beetle luciferin in a reaction that requires ATP, Mg2+ and O2 (FIG. 1).
Renilla luciferase is a 36 kDa monomeric protein that is composed of 3% carbohydrate when purified from its natural source, Renilla reniformis. Like firefly luciferase, post-translational modification of Renilla luciferase is not required for its activity, and it functions as a genetic reporter immediately following translation. The luminescent reaction catalyzed by Renilla luciferase utilizes O2 and coelenterate-luciferin, also called coelenterazine (FIG. 2).
Luminescent reactions can be used to detect very small quantities of a particular analyte, the substance being identified and measured in an analysis. For example, luminescent reactions can be used to detect and quantify proteases, lipases, phosphatases, peroxidases, glycosidases, and various metabolites such as ATP or NADH. Luminescent reactions can also be used to detect and quantify analytes through binding interactions, such as those mediated by antibodies and nucleotide probes. Typically, luminescent reactions can be used to detect less than 1×10−16 moles of analyte in a sample, often less than 1×10−19 moles. In luminescence, commonly detected analytes are the luciferases, especially firefly luciferase and Renilla luciferase. Most often these analytes are used to quantify phenomena associated with their creation through gene expression and protein synthesis. Other luminescent enzymes used as analytes include, but are not limited to, aequorin, Vargula luciferase, and other marine luciferases.
When using luminescence to measure an analyte, it is preferred that little or no light is produced by reactions that are not dependent on the presence of the analyte. This is the case with firefly luciferase. Under typical firefly luciferase assay conditions, luminescence cannot be detected when the firefly luciferase is not present. In contrast to assays employing firefly luciferase, light can generally be detected in Renilla luciferase assay systems when the Renilla luciferase is not present. Luminescence that is not dependent on the catalytic activity of a luminescent enzyme is termed autoluminescence. For example, autoluminescence can be caused by spontaneous oxidation of the luminogenic substrate coelenterazine.
Luminescence that is not dependent on the on the presence of an analyte (e.g. autoluminescence) can limit the usefulness of an analytical assay by reducing the ability to accurately measure the quantity of light resulting from the activity of the analyte. In particular, the sensitivity of luminescent assays containing coelenterazine or its structural analogs is reduced due to autoluminescence. Additionally, the addition of various components to the assay system, such as lipids (especially above the critical micelle concentration or CMC), hydrophobic proteins (especially those with a defined three-dimensional structure), and cells or other biological materials containing hydrophobic microenvironments, can greatly increase autoluminescence.
Assay sensitivity may also be reduced by luminescence from an unrelated luminogenic molecule. The unrelated luminogenic molecule may be present due to contamination of the analytical assay, or due to a separate analytical luminescence assay performed in the same reaction mixture. In either case, the sensitivity of an analytical luminescence assay could be improved by reducing the luminescence that is not dependent on the presence of the analyte.